The invention relates to analyte-binding assays, such as iron binding assays.
Serum total iron binding capacity (TIBC) can be used to assess a patient""s iron metabolic state. Typically, at least about 95% of iron in serum is bound by transferrin that binds ferric iron with an anion (preferably bicarbonate) at pH greater than about 5. Often, only about ⅓ of the transferrin serum binding sites are occupied with iron.
The invention relates to analyte-binding assays, such as iron binding assays.
In one aspect, the invention generally features a method of evaluating a sample. The sample includes an analyte-binding compound and an analyte capable of binding to the analyte-binding compound to form an analyte-compound complex. The method includes combining the sample with an analyte-binding dye to form a first mixture. The analyte-binding dye is capable of binding to the analyte to form an analyte-dye complex. The method also includes comparing a measurement of the amount of the analyte-dye complex in the first mixture taken under a first condition to a measurement of the amount of the analyte-dye complex in a second mixture that includes the first mixture taken under a different condition. The affinity of the analyte-binding compound for the analyte under the first condition is different from the affinity of the analyte-binding compound under the second condition (e.g., the affinity of the analyte-binding compound for the analyte under the first condition is less than the affinity of the analyte-binding compound for the analyte under the second condition).
The measurements can be made by evaluating the ability of the analyte-dye complex to interact with energy (e.g., by measuring absorbance, emission and/or reflectivity).
The first condition can be one pH and the second condition can be a different pH. For example, the first pH can be lower than the second pH.
The analyte can be iron ions (e.g., ferric ions), the analyte-binding dye can be an iron-binding dye, and the analyte-binding compound can be transferrin.
The method can take less than about 45 minutes.
The method can include adding a base to the first mixture to form the second mixture.
In some embodiments, less than about 5% of the analyte in the first mixture is bound to the analyte-binding compound in the first mixture under the first condition.
The first mixture can include sufficient analyte so that substantially all the analyte-binding compound is saturated with the analyte in the second mixture, and the method can be performed without further addition of analyte.
The difference between the first and second measurements can be proportional to the total iron binding capacity of the sample.
In another aspect, the invention generally features a method of evaluating a sample containing ferric ions and transferrin. The method includes combining the sample with an iron-binding dye to form a first mixture. The iron-binding dye is capable of binding ferric ions to form an iron-dye complex. The method also includes comparing a measurement of the amount of the iron-dye complex in the first mixture to a measurement of the amount of the iron-dye complex in a second mixture that includes the first mixture. The first measurement is made at one pH (e.g., less than about 5.7) and the second measurement is made at a higher pH (e.g., greater than about 5.7).
The iron-binding dye can be a fluorescent dye and/or a colorimetric dye. For example, the iron-binding dye can be Eriochromcyanine R, Chromazurol S and/or Chromazurol B.
The first mixture can contain excess ferric ions and/or excess iron-binding dye.
In some embodiments, less than about 5% of the ferric ions in the first mixture are bound to transferrin at the first pH.
The first mixture can contain a sufficient amount of ferric ions so that substantially all the transferrin is saturated with ferric ions in the second mixture.
In certain embodiments, the difference between the first and second measurements is proportional to the total iron binding capacity of the sample.
In a further aspect, the invention generally relates to a method of determining the amount of iron in a sample and the total iron binding capacity of the sample. The method includes comparing a measurement of the amount of an iron-dye complex in a first mixture, which contains the sample, to a measurement of the saturating amount of the iron-dye complex in the absence of the sample to determine the amount of iron in the sample. The mixture is measured when under a first condition. The method also includes comparing a measurement of the amount of the iron-dye complex in a second mixture, which contains the first mixture, to the measurement of the iron-dye complex in the first mixture to determine the total iron binding capacity of the sample. The second measurement of iron-dye complex in the second mixture is made when the second mixture is under a second condition that is different from the first condition.
The method can further include combining the sample with a composition to form the first mixture. The composition can contain a sufficient amount of the iron and/or iron-dye complex so that first mixture contains the saturating amount of iron and/or the iron-dye complex.
The sample can contain ferric ions and transferrin.
The first condition can be one pH, and the second condition can be a different pH. For example, the first condition can be a lower pH than the second condition.
The method can include adding a base to the first mixture to form the second mixture.
The first mixture can contain a sufficient amount ferric ions so that substantially all the transferrin contained in the second mixture is saturated with ferric ions.
The sample can be a serum sample.
In another aspect, the invention generally relates to a method of determining the amount of an analyte in a sample and the amount of an analyte-binding compound in the sample. The analyte-binding compound is capable of binding the analyte to form an analyte-compound complex. The method includes comparing a first measurement of the amount of an analyte-dye complex in a first mixture, which contains the sample, to a measurement of the saturating amount of the analyte-dye complex in the absence of the sample to determine the amount of the analyte in the sample. The first measurement is made when the mixture is under a first condition. The method also includes comparing a second measurement of the amount of the analyte-dye complex in a second mixture, which contains the first mixture, to the first measurement of the analyte-dye complex in the first mixture to determine the amount of analyte-binding compound in the sample. The second measurement of analyte-dye complex in the second mixture is made when the second mixture is under a second condition different from the first condition.
In another aspect, the invention includes a reagent discussed herein or a kit including one or more reagent described herein and optionally one or more of calibration standards and instructions.
The assay can provide the advantage of not requiring a separate step in which free iron is isolated from bound iron. This can reduce the time and cost associated with the assay relative to other iron binding assays which involve a separate step in which free iron is isolated from bound iron.
The invention can be advantageous because it can reduce and/or eliminate biases that can exist between commercially available methods of indirectly making TIBC measurements and other conventional methods.
The invention can be advantageous because the assay can reduce sample manipulation and/or allow determination of serum iron and TIBC in a single assay.
The invention can be advantageous because the assay can be conducted under conditions where the saturating iron present in the reagent can be more stable as part of the analyte-dye complex.
One potential advantage of the invention is that the assay can be conducted in a relatively short period of time.
Another potential advantage of the invention is that the assay can be conducted in a single vessel (e.g., without using multiple vessels).
A further potential advantage of the invention is that the assay can be conducted without removing the analyte from the mixture (e.g., without removing the analyte from the mixture under the first condition or under the second condition).
Another potential advantage of the invention is that it can provide methods of measuring the percent analyte saturation (e.g., percent iron saturation) of a sample in a single process (e.g., using one reaction vessel, without removing the analyte from the mixture and/or without isolating the analyte from the mixture).
Other features and advantages of the invention will be apparent from the figures, the following detailed description, and from the claims.